Lithospermum erythrorhizon that has been proved to possess powerful anti-proliferative effect on many kinds of cancers and to be safe in in vivo study. Posterior capsular opacification (PCO), the most frequent complication of cataract surgery, is mainly caused by the uncontrolled proliferation of retained human lens epithelial cells (HLEs). In this study, we investigated the effect of shikonin on the proliferation of HLEs and explored its underlying mechanism of action. Shikonin significantly inhibited the proliferation of HLEs in a dose- and time-dependent manner. Its anti-proliferative effect was exerted through induction of apoptosis. Reactive oxygen species (ROS) generation played an essential role in this apoptotic process. Interestingly, scavenging of ROS completely blocked the apoptosis induced by shikonin. In addition, the treatment of shikonin in HLEs significantly increased the ratio of Bax/Bcl-2, disrupted mitochondria membrane potential (MMP) and activated caspases. The inhibition of caspase largely blocks the apoptosis. The changes of MAPK pathway were also demonstrated. Shikonin effectively inhibited the phosphorylation of ERK, while it activated the phosphorylation of JNK and p38. These results suggested that shikonin inhibited the proliferation of HLEs by inducing apoptosis through ROS generation and the caspase-dependent pathway and the MAPK pathway was also involved.Shikonin is a compound from the herbal plant  The preLithospermum erythrorhizon, and it has been widely used in China for centuries for its anti-inflammation properties and the treatment of external wounds [Shikonin is a naphthoquinone derived from the root of l wounds . The resl wounds . It has l wounds ,16,17, b2DCFDA  respectively.In this study, we examined anti-proliferative effect of shikonin on HLEs and attempted to explore its underlying mechanism of action. The changes of cells in apoptosis, MMP and ROS production were examined by flow cytometry after staining with Annexin V/propidium iodide (PI), JC-1  and CM-H50 value of shikonin on HLE B-3 at 24, 48 and 72 h is 4.58 ± 0.76 μM, 2.47 ± 0.52 μM and 1.86 ± 0.33 μM respectively. These suggested that shikonin significantly inhibited the proliferation of HLE B-3 cells.To investigate the anti-proliferative effect of shikonin on HLEs, HLE B-3 cells were exposed to different concentrations of shikonin for 24, 48 and 72 h. Then the inhibition of proliferation was determined by an MTT assay. As shown in To investigate whether shikonin exerts its inhibitory effect by inducing apoptosis in HLEs, an Annexin V/PI double staining assay was applied. Annexin V was used to detect phosphatidylserine externalization, which is a hallmark of the early phase of apoptosis. PI on the other hand is a label of DNA fragments, a symbol of cell death. As shown in To determine whether shikonin-induced apoptosis is through a mitochondria dependent pathway, the MMP of cells was detected with the mitochondria-sensitive dye, JC-1 . As showMoreover, the activation of caspases was also detected. As shown in 2DCFDA, a general oxidative stress indicator. As shown in To elucidate whether shikonin-induced apoptosis on HLE B-3 cells is due to oxidative stress, cells were exposed to different concentration of shikonin for 2 h and the level of ROS was measured by using CM-HFurthermore, we tried to demonstrate whether ROS plays an essential role in the anti-proliferation effect of shikonin in HLEs. NAC, a specific inhibitor of ROS was applied as a co-treatment with shikonin. From It has been extensively proved that the mitogen-activated protein kinases (MAPK) pathway plays a critical role in controlling cell proliferation and death. It has been reported that shikonin can regulate JNK and p38 activation in chronic myelocytic leukemia . To deteN-acetyl-L-cysteine (NAC) were purchased from Sigma Aldrich . Pan-caspase inhibitor was purchased from BD Pharmingen . Antibodies against Bcl-2 and Bax were purchased from Santa Cruz Biotechnology . And antibodies of caspase-3/7, phospho-ERK, total ERK, phospho-JNK, total JNK, phospho-p38, total p38, PARP, were purchased from Cell Signaling Technology . The fluorescein-conjugated secondary antibodies were purchased from Odyssey .Shikonin powder and 2 [HLE B-3 was purchased from ATCC and was the only human lens epithelium cell line which ATCC provided. Cells were cultured with RPMI 1640 medium supplemented with 20% fetal bovine serum , 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco), and cultivated at 37 °C with an incubator supplying 5% CO2 .HLE B-3 cells were seeded on a 96 well microplate with 5000 cells/well, and were cultured overnight for cell adhesion. Shikonin was added and was incubated for 24, 48 or 72 h with vehicle Dimethyl sulfoxide (DMSO) as the control. Each dosage was repeated as triplicate. Ten μL of MTT (5 mg/mL) was added to every well and the plate was placed in the incubator for 4 h. Then 100 μL of formazan crystals solubilizing solution (10% SDS and 0.01%M HCL) was added to each well and the plate was further incubated at 37 °C for another 4 h to dissolve the formazan crystals. Finally, after shaking 10 min at room temperature, the absorbance of each well was measured at 570 nm using a microplate reader . The cell viability is calculated by dividing the absorbance of treated cells by that of vehicle control.According to the manufacturer’s (BD Pharmingen) protocol, cells were harvested, washed, and double-stained with Annexin V/PI for 15 min at room temperature in dark. Then apoptotic cells were quantitatively counted by a flowcytometry. Early stage of apoptotic cells were stained as Annexin V-positive and PI-negative, while the late stage apoptotic cells were stained as Annexin V-positive and PI-positive.JC-1 staining (BD Pharmingen) was applied to determine the MMP level of cells. After treatment, cells were harvested, washed, and stained with JC-1 for 15 min in the incubator at 37 °C. Then cells were washed again and re-suspended with regular medium. The change in MMP of the cells was measured by flow cytometer followed with the manufacturer’s protocol. In brief, the fluorescence emission spectrum of JC-1 is dependent on the concentration of the JC-1 forms. There are two forms of JC-1 which include the monomer and the aggregate forms. The monomer form of JC-1 represents the loss of MMP and yields a green fluorescence signal, while the increase of red fluorescence indicates that JC-1 aggregates together in normal hyperpolarized mitochondria.® 20 (TBST) three times (5 min/time), secondary fluorescent antibody (1:10000 dilutions) was added to membrane at room temperature for 1 h. Actin was used as loading control and for normalization. The signal intensity of the membranes was detected by Odessy .After treatment, ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer with protease and phosphatase inhibitors was add to the cells for total cellular protein extraction. The concentration of the total cell lysate was determined by Bio-Rad detergent-compatible (DC™) colorimetric protein assay kit and proteins were denatured by heating 95 °C for 5 min. The prepared samples were loaded onto a 10% SDS-PAGE gel to separate. After electrophoresis, the proteins on SDS-PAGE gel were transferred to a Nitrocellulose (NC) membrane at 300 mA for 2 h. Membranes were blocked with 5% milk without fat in TBST for 1 h at room temperature. Primary antibodies  were incubated overnight at 4 °C. After washing by Tris Buffered Saline with Tween2DCFDA, Invitrogen, Carlsbad, CA, USA) fluorescent probe staining, which is a specific superoxide tracing dye. Cells were pretreated with CM-H2DCFDA 20 μM 30 min at 37 °C prior to treatment. And then cells were incubated for the indicated times with shikonin and vehicle control, harvested and re-suspended in phosphate buffered saline (PBS). Fluorescence was measured with flowcytometry with excitation and emission settings of 488 and 525 nm, respectively.ROS was analyzed by using dichlorofluorescein diacetate  of three individual experiments. Differences between groups were determined by one way analysis of variance (ANOVA), followed by Dunnet’s test to compare the treated groups with the control, with After cataract surgery, the residual HLEs at the equator and under the anterior lens capsule proliferate and migrate onto the posterior capsule and undergo epithelial-mesenchymal transition, resulting in the formation of fibroblasts, myofibroblasts, and extracellular cell matrix, and finally, PCO .PCO is the most frequent complication and the primary reason of visual decrease after surgery . AlthougLithospermum erythrorhizon, a herbal medicine with various biological activities [in vivo [Shikonin is a major component of tivities . It has [in vivo . In this[in vivo .In this apoptotic process, ROS generation was remarkably up-regulated and played a necessary role. Once the cells fail to remove the excessive ROS, this will lead to mitochondrial disruption and cell death, which was proved by an MMP assay. During mitochondrial dysfunction, Bcl-2 family members cause outer membrane permeabilization, which subsequently allows cytochrome C that is sequestered in the mitochondrial intermembrane space to be released into the cytosol and activate caspases . We founin vivo [The MAPK superfamily consists of three main protein kinase families: ERKs, JNK and the p38. It has been reported that they are all closely correlated with the growth of cells and plays an essential role in regulating the intracellular metabolism and responding to external stress . It has in vivo . Moreovein vivo , which cin vitro study is only preliminary. Further detailed and intensive researches in vivo and even the clinic should be undertaken to elucidate the benefits and effects of shikonin on PCO.In sum, we have demonstrated that shikonin-induced apoptosis occurs through ROS generation and a caspase activation dependent pathway. The MAPK pathway was also involved. This may provide a new avenue for treating PCO patients in the future. However, the present


Streptomyces lividans has been used as a host for engineering extracellular overproduction of homologous and heterologous proteins in industrial applications. Although the mechanisms governing the major secretory pathway (Sec route) and the minor secretory pathway (Tat route) are reasonably well described, the function of proteins responsible for the extracellular secretory protein folding is not characterized as yet. We have characterized a Tat-dependent S. lividans FK506-binding protein-like lipoprotein (FKBP) that has PPIase activity. A mutant in the sli-fkbp gene induces a secretion stress response and affects secretion and activity of the Sec-dependent protein α-amylase. Additionally, propagation in high copy number of the sli-fkbp gene has a positive effect on the activity of both the overproduced α-amylase and the overproduced Tat-dependent agarase, both containing proline cis isomers. Targeted proteomic analyses showed that a relevant group of secreted proteins in S. lividans TK21 are affected by Sli-FKBP, revealing a wide substrate range. The results obtained indicate that, regardless of the secretory route used by proteins in S. lividans, adjusting the expression of sli-fkbp may facilitate folding of dependent proteins when engineering Streptomyces strains for the overproduction of homologous or heterologous secretory proteins.Some bacterial peptidyl-prolyl  The genome sequence of S. lividans is known [is known ,4, and tis known ,6.S. lividans cytoplasmic membrane mainly uses the major secretion pathway (Sec) and the minor twin arginine translocation (Tat) secretion pathway [Streptomyces coelicolor Tat system [S. lividans, the Tat system has three components , and the Tat signal peptide, containing the highly conserved twin-arginine motif SRRXFLK, at its amino end, is thought to be recognized by the TatA–TatB heterocomplex [Escherichia coli the signal peptide recognition is mediated by the TatB–TatC complex [Extracellular protein secretion across the  pathway . The Sec pathway . The rib pathway . Thus, tt system . In S. locomplex . This coocomplex . In Esch complex .cis/trans isomerases (PPIases). Bacterial PPIases have been linked to protein folding and secretion because of their capacity to catalyse the cis/trans isomerization of peptide bonds preceding prolyl residues [The main enzymes involved in extracellular folding of secretory proteins are the thiol-disulfide oxidoreductases and the peptidyl-prolyl residues . The PPIresidues .Bacillus subtilis PrsA is a lipoprotein that belongs to the parvulin family and is considered an important factor for protein secretion. The cellular level of PrsA and the rate of secretion have been shown to be interrelated in B. subtilis. Depletion of PrsA leads to a reduction of the levels of secreted heterologous α-amylase (AmyQ) [e (AmyQ)  as well e (AmyQ) . PrsA ove (AmyQ) .S. lividans yet. In the present work, we characterize a Tat-dependent S. lividans FKBP-like lipoprotein (Sli-FKBP) that positively affects the production of a significant number of Sec- and Tat-secreted proteins containing putative proline cis isomers in S. lividans TK21.No equivalent PPIases have been experimentally characterized to exert this role in 2.2.1.S. lividans TK21 wild-type strain [−1), thiostrepton (50 µg ml−1), kanamycin (50 µg ml−1) and chloramphenicol (25 µg ml−1) were added to the R5 and MS solid media, when required.The e strain  and its e strain . Apramyc2.2.S. lividans Δsli-fkbp mutant strain oligonucleotides FKBPdisFw (5′-GGGCTGCAGAACAGCTACGACCGCAAGAC-3′) and FKBPdisRv (5′-GCCTCTAGACACACCCTTGTACTGCACGA-3′) were used to amplify a 420 nt long DNA fragment that was inserted into the non-replicating suicide plasmid pOJ260 of Streptomyces [XbaI and PstI sites to generate plasmid pOJFKBP. The plasmid was used to conjugate E. coli to Streptomyces as described [sli-fkbp by insertion of pOJ260 by single homologous recombination between pOJFKBP and the sli-fkbp copy in the chromosome. Escherichia coli ET12567 carrying the non-transmissible ‘driver’ plasmid pUZ8002 was used for conjugation [sli-fkbp were selected upon verification of the disruption by polymerase chain reaction (PCR) amplification (not shown). Plasmids pAMI11 [S. lividans gene amlB or the S. coelicolor agarase gene dagA, respectively, were used to transform the S. lividans TK21 and S. lividans Δsli-fkbp.To construct the ptomyces  through jugation . Apramycs pAMI11  and pAGAs pAMI11  carrying2.3.HindIII and XbaI sites and FKBP-P (5′-TTTGGATCCTTCTCACGCCGTAGAGTTGC-3′) and FKBP-T (5′-TTTTTGGATCCCCCTCCCAGATGTCCTTGAT-3′) containing the BamHI sites were used to amplify the gene sli-fkbp and its possible promoter region. Chromosomal DNA of S. lividans TK21 strain was used as a template. The obtained 1222 bp long DNA fragments were subsequently cloned into the pGEM-T Easy vector (Promega) and sequenced. The plasmid pGEM, harbouring sli-fkbp, was digested with NotI to retrieve the 1244 bp long DNA fragment containing sli-fkbp; this was inserted into the multi-copy cosmid pFDT [NotI site to generate pFDTFKBP. Additionally, the pGEM harbouring sli-fkbp was digested with BamHI, and the 1222 bp long DNA fragment was cloned into the multicopy plasmid pIJ487 previously digested with BamHI to generate plasmid pIJFKBP.The oligonucleotides FKBP-HindIII (5′-TTTTAAGCTTTCTCACGCCGTAGAGTTGC-3′) and FKBP-XbaI (5′-TTTTTCTAGACCCTCCCAGATGTCCTTGAT-3′) containing the S. lividans TK21, S. lividans TK21(pAMI11) and S. lividans TK21(pAGAs5) were transformed with cosmid pFDTFKBP to obtain the S. lividans (pFDTFKBP), S. lividans TK21(pAMI11) (pFDTFKBP) and S. lividans TK21(pAGAs5) (pFDTFKBP) strains. Cosmid pFDT was propagated in S. lividans TK21, S. lividans TK21(pAMI11) or S. lividans TK21(pAGAs5) to generate the corresponding isogenic strains.Protoplasts from S. lividans TK21 were transformed with plasmid pIJFKBP to obtain S. lividans (pIJFKBP). Plasmid pIJ487 was propagated in S. lividans TK21 to generate the corresponding isogenic strain.Protoplasts from 2.4.Total RNA was isolated from bacteria growing cultures at different phases of growth  using the RNeasy midi Kit (Qiagen). Cell lysates were extracted twice with phenol–chloroform before being loaded onto RNeasy midi-columns for RNA purification. DNA, potentially contaminating the RNA preparations, was removed by incubation with RNase-free DNAse (Ambion) and its absence was tested by quantitative real-time PCR (qRT-PCR) amplification in the absence of reverse transcriptase. Complementary DNA was synthesized using the High Capacity Archive kit (Applied Biosystems). qRT-PCR was performed using SYBR Green technology in an ABI Prism 7300 Sequence Detection System (Applied Biosystems). Samples were initially denatured by heating at 95°C for 10 min. A 40-cycle amplification and quantification programme was then followed (95°C for 15 s and 60°C for 1 min) by a single fluorescence measurement per cycle, according to the manufacturer's recommendations. Subsequently, a final extension cycle  was performed. Three biological samples from the different bacterial cultures were amplified in triplicate in separate PCR reactions. All PCR products were between 50 and 150 bp in length.hrdB transcript was carried out as an internal control to quantify the relative expression of the target genes. The hrdB transcript was used as a reference to normalize the relative expression of Streptomyces genes. The oligonucleotides used as primers to amplify the transcripts of two-component system cssRS and tatC genes were described previously [sli-fkbp transcript.A melting curve analysis was conducted after amplification to distinguish the targeted PCR products from the non-targeted ones. The melting curves were obtained by heating at temperatures ranging from 60°C to 95°C at a rate of 0.2°C per second, with continuous fluorescence scanning. The eviously ,26. Olig2.5.sli-fkbp mutant and the Sli-FKBP overproducer strains containing the multi-copy plasmids pAMI11 and pAGAs5 grown in NMMP medium [Supernatants from the P medium  were proP medium . For wesP medium . An equiS. lividans TK21 AmlB (a gift from C. Isiegas) and S. coelicolor agarase , as described . The trae  followee .To determine extracellular α-amylase activity and agarase activity, the supernatants from the aliquots of bacterial cell cultures were collected at the indicated growth phases and concentrated by precipitation with ammonium sulfate brought to 80% saturation; the precipitated protein was collected by centrifugation at 13 000The activities of α-amylase and agarase were estimated by determining the amount of reducing sugars released from starch and agarose, respectively. α-amylase and agarase activities were determined as previously described ,30. One One unit of agarase activity is the amount of enzyme that increased absorbance at 450 nm by 0.001 per minute of incubation under the assay conditions. The specific activity was expressed as units per mg of dry weight and was the average of at least three independent determinations.−1 lysozyme. After 20 min of incubation at 37°C protoplasts were washed with P buffer and then were centrifuged at 1500g for 5 min at 4°C. The protoplast's pellet was then re-suspended in 300 µl P buffer, and 30 µl aliquots were used to perform the assay.The FKBP activity was determined using protoplasts. Protoplasts were prepared as previously described . BrieflyN-succinyl-L-Ala-Ala-Pro-Phe-p-nitroanilide (sAAPF-pNA) (Sigma Aldrich ref. S7388). Chymotrypsin cleaves the synthetic peptide only when the Ala-Pro bond, which is in equilibrium between cis and trans, is in trans configuration [p-nitroanilide) with a spectrophotometer . FK-506 (Sigma Aldrich ref. F4679) was added in the inhibition of the PPIase activity assays [cis/trans isomerization of X-Pro peptide bonds. Therefore, the isomerization reaction of this peptide bond was measured by monitoring the release of p-nitroaniline.The isomerase assay was determined by protease coupling assay with α-chymotrypsin (α-Ct) (Sigma Aldrich ref. C4129) using as a substrate guration . The reay assays . PPIase −1 dissolved in dimethyl sulfoxide were added and incubated for 3 min on ice before the absorbance was measured at 390 nm for 1–6 min. For the inhibition assay 2 µl of FK506 14.2 µM dissolved in 50% (v/v) aqueous ethanol was added to the 30 µl aliquots and 625 µl of 110 mM Tris HCl pH 8 and then incubated for 15 min on ice. After that, chymotrypsin and sAAPF-pNA were added [Aliquots (30 µl) of the pIJ487 and pIJFKBP protoplasts were mixed with 625 µl of 110 mM Tris HCl pH 8 and water to a total volume of 710 µl and incubated for 15 min on ice. Then, 120 µl of α-Ct 0.56 mM dissolved in 1 mM HCl and 30 µl of sAAPF-pNA 1.4 mg mlre added . The res2.7.cis-trans prolyl isomers [For the prediction of the signal peptide SignalP 4.1, LipoP 1.0 and TatP 1.0 servers –35 were  isomers .2.8.trans conformation were generated using UCSF Chimera [Full three-dimensional protein models were generated using I-TASSER , Modelle Chimera  and subj Chimera  and the  Chimera  force fi Chimera .2.9.S. lividans (pAGAs5) (pFDTFKBP) extracellular protein band reacting positively with anti-agarase antibodies was sliced out of the SDS-PAGE and digested with trypsin using Proteineer DP robot . Digestion was performed according to a previously described protocol [For protein identification, the protocol . In summ−1, in 0.1% formic acid in water. The trap column was switched online to a C18 nanoAcquity BEH analytical column . Chromatographic elution was achieved using a 40 min linear gradient ranging from 5% to 40% solvent B (0.1% formic acid in acetonitrile) at 250 nl min−1. The mass spectrometer operated in data-dependent acquisition mode. For TOF scans, the accumulation time was set to 250 ms, and 15 precursor ions were monitored per cycle.The peptide samples were analysed on a nano-liquid chromatography (LC) system  coupled to a 5600 TripleTOF mass spectrometer  with a nanoelectrospray ion source. Samples were loaded on a C18 PepMap trap column  at 2 µl minS. coelicolor at Uniprot Knowledgebase (as of November 2016), using a licensed Mascot Server v. 2.4 search engine . Search parameters were set as follows: carbamidomethylation of cysteine as fixed modification and methionine oxidation as variable 1. Peptide mass tolerance was set to 25 ppm and 0.02 Da, in MS and MS/MS mode, respectively and one missed cleavage was allowed.Mass spectrometry (MS) and tandem mass spectrometry (MS/MS) spectra were processed using Analyst TF 1.5.1 software . Raw data were converted to MGFs  and searched against a database built from sequences obtained for 2.10.n = 36) group of proteins was determined by targeted proteomics, using selected reaction monitoring (SRM/MRM). For that purpose, 1 mg of trypsin-digested proteins per sample was analysed using an Eksigent 1D Plus nano-LC system coupled to a SCIEX 55000 QTRAP quadrupole triple mass spectrometer. Using a 45 min length gradient, 273 transitions that corresponded to 83 specific peptides of the 36 proteins of interest were monitored. Protein-specific peptides and transitions were selected, whenever possible, on the basis of previous experimental data. As the number of transitions was too high to analyse using a single SRM/MRM method, two sub-methods were designed. Nine samples  were monitored using both sub-methods and the raw data files in WIFF format were analysed using the Skyline 4.2 software [2-transformed to approach normality and comparisons between different strains were performed using Student's t-test and Fisher's exact test.Relative protein abundance of a selected  having a PPIase activity [S. lividans TK24 gene SLIV_29545 has been shown to be highly homologous to the SCO1639.Analysis of the  protein . This ge protein  that is activity . The S. S. lividans TK21 gene homologous to the SLIV_29545 gene could also encode a PPIase with a potential role in the correct folding of secretory proteins, oligonucleotides FKBP-P and FKBP-T were used to amplify a 1222 bp long DNA fragment from the S. lividans genome containing the DNA sequence encoding the putative FKBP-like protein and the potential regulatory region of the gene.To test if the equivalent S. coelicolor and the S. lividans TK24 genomes and found to coincide in 99% and 100%, respectively, with that of the genes encoding their respective FKBP-like proteins. The FKBP-like protein encoded in S. lividans TK21 was predicted to be a substrate for a type II signal peptidase [S. lividans TK21 FKBP-like protein was found to be lower in an S. lividans TK21 strain defective in the type II signal peptidase Lsp [The 1222 bp long amplified DNA fragment was sequenced and its sequence was aligned with the equivalent ones from the eptidase . Moreovedase Lsp , when me3.2.S. lividans FKBP-like protein showed 36% identical residues and 50% equivalent amino acids to the E. coli periplasmic FKBP (FkpA), which acts as a periplasmic folding modulator with a broad substrate range [The te range . Activitte range .S. lividans FKBP-like protein is a lipoprotein, to perform the PPIase activity assay we used protoplast extracts of the S. lividans pIJ486 and S. lividans pIJFKBP strains, containing the multi-copy plasmid pIJ486 and the multi-copy plasmid pIJ486 harbouring the fkbp-like gene, respectively (p-nitroaniline released from the substrate upon incubation in the absence of protoplasts were subtracted from those obtained in the presence of pIJ486 protoplasts and pIJFKBP protoplasts. A higher increase in the absorbance was observed for protoplasts containing pIJFKBP owing to the presence of an excess of FKBP copies versus the protoplast containing pIJ487 at 120 and 240 s (a).Since ectively . The bacnd 240 s a.Figureb). From now on we will refer to the S. lividans TK21 gene encoding the FKBP-like protein as sli-fkbp.The presence of the inhibitor FK506 decreases the absorbance to 0 at 120 s and recovers the absorbance observed with respect to the control sample (without inhibitor) at 240 s in the pIJ487 protoplasts, which represents a percentage of inhibition of 100% at 120 s. The values of residual activity after treatment with the inhibitor FK506 in the pIJFKBP protoplasts was 0%, 30% and 76.23%, which represent a percentage of inhibition of 100%, 70% and 23.77% at 120, 240 and 360 s, respectively b. From n3.3.hrdB gene, showed that the expression of the sli-fkbp and tatC in the S. lividans TK21 strain runs parallel to cell culture growth, although the level of the sli-fkbp expression is lower than that of the tatC gene (about 10% that of tatC) [sli-fkbp expression, the overall amount of misfolded secreted proteins would likely increase and, consequently, the level of expression of the two-component CssRS system [sli-fkbp disrupted mutant (sli-fkbp::pOJ260) was constructed, and the relative level of expression of the CssRS two-component system genes was determined by qRT-PCR analyses and compared with that of the wild-type cell. A significant increase in the expression levels of the two genes (relative expression levels of the sensor cssS: 25.02 ± 1.35 and of the regulator cssR: 8.35 ± 1.11) was observed at the late exponential phase of growth (24 h), suggesting that the absence of the sli-fkbp gene may produce an accumulation of misfolded proteins at this time. This suggests that, despite the moderate level of expression of the single sli-fkbp copy present in the bacterial genome, the Sli-FKBP protein may play an active role in the correct folding of extracellular proteins.The qRT-PCR transcriptional analyses, using as an endogenous control the of tatC) . In the S system ,25 could3.4.cis conformation at residue 350 (P350) may be required to attain the mature form of the Sec-dependent protein α-amylase. Similarly, the Tat-dependent protein agarase may require prolyl isomerization at residues 127 and 183 to the cis conformation to produce mature active agarase .Prolyl isomerization to the S. lividans, we propagated the multi-copy plasmids pAMI11 and pAGAs5 harbouring the S. lividans α-amylase gene (amlB) and the S. coelicolor agarase gene (dagA), respectively, in the sli-fkbp mutant and determined the secretion and extracellular activities of α-amylase and agarase.To explore further the role of Sli-FKBP in the secretion of proteins in amlB and dagA in the sli-fkbp mutant strain constrained their growth rate when compared with the wild-type . The Sec-dependent protein α-amylase reaches its maximum level of secretion at 24 h of growth while agarase reaches its maximum level of secretion during the stationary phase [a,c). The secretion pattern and extracellular activity of α-amylase were severely affected in the sli-fkbp mutant strain with respect to that of the isogenic strain , showing the presence of inactive α-amylase only at 60 h and probably corresponding to incorrectly folded protein as previously described [c,d).Overexpression of ry phase   strain.To study if overproduction of Sli-FKBP could increase the production of the Sec-dependent model protein α-amylase, the pFDTFKBP plasmid (a plasmid compatible with pIJ486) was propagated in the a,b), suggesting that secreted α-amylase may benefit from the action of Sli-FKBP for correct folding. Moreover, the level of expression of the CssRS two-component system genes decreased in the strain that overproduces α-amylase and sli-fkbp (relative expression levels of the sensor: cssS −2.42 ± 0.113 and of the regulator cssR: −1.79 ± 0.89) versus the strain lacking multi-copy sli-fkbp, where the two-component system was activated [Overproduction of Sli-FKBP did not affect significantly the rate of growth in liquid medium . α-amylase secretion increased, with an increase in extracellular activity of around threefold with respect to the isogenic strain overproducing α-amylase a,b, suggctivated . This su. lividans (pAGAs5) strain. Extracellular agarase activity registered a threefold increase in the strain overexpressing agarase and sli-fkbp (d), suggesting that overproduction of Tat-dependent agarase needs additional activity of Sli-FKBP to fold correctly. No significant increase was detected in the amount of secreted agarase by western blot assays (c). Incidentally, the western blot analyses showed a difference between strains in the gel mobility of the protein produced (c). The faster moving band, corresponding to agarase and Sli-FKBP overproduction, was sliced out of the gel and the protein analysed by nano LC-MS/MS triple TOF MS. The proteomic analysis allowed recovery and identification of tryptic peptides of mature agarase, obtaining 65% coverage of the overall protein , which would correspond to 20 kDa according to ProtParam [To test if higher levels of FKBP would be needed to improve the extracellular activity of secreted agarase, the multi-copy plasmid pFDTFKBP was propagated in the Ssli-fkbp d, suggest assays c. Incideproduced c. The farotParam . This sirotParam ,56, and 3.6.cis peptide bonds, at P127 and P183, equivalent to those found on other homologues. Switching of P127 to the trans conformation would disrupt the allosteric agarose-binding site, preserving the active site, while switching of P183 would disrupt both . The trans forms show a conformation with lower potential energy (more stability) than the cis structures. However, the mature double-cis form contains numerous H-bonds that help stabilize it. This suggests that the spontaneous change from trans to cis would be energetically unfavourable, justifying the need for a PPIase to facilitate it, while the H-bond network in the cis form would provide a significant initial barrier to spontaneous reversion to the trans conformation afterwards.The best agarase models were selected by inspecting the homology models obtained with various methods according to coherence with published structures and available biological information. MD simulations of the chosen models revealed the existence of one preferred conformation that was used as a representative for further analysis. The predicted structure contains two trans forms would have larger molecular dimensions , and the distribution of surface charges would also be different: conversion to cis would be associated with a larger reduction (greater than 400 Å2) of surface positive charge than of the surface negative charge. Both of these effects (changes in dimension and surface charge) could contribute to the differential mobility of the cis and trans forms.In addition, the 3.7.sli-fkbp, the single-copy sli-fkbp and the sli-fkbp overexpressing strains using three biological replicates.To gain further insight into the effect of the overproduction of Sli-FKBP on secreted proteins we performed an SRM/MRM proteomic experiment. SRM/MRM is typically used to perform reliable, sensitive and selective protein assays for the comparison of relative protein amounts between samples . The anaS. lividans TK21 were analysed with the SPOT-Omega server [cis–trans isomers. A group of 31 secreted proteins that might be potential substrates of Sli-FKBP  were selected for further analysis using SRM/MRM. Sli-FKBP was also included in the analysis to compare its expression in the three strains.Cognate secreted proteins previously identified in the supernatant of a server  to identsli-fkbp strain but were detected in the other strains. The ratios and significance level  of differences in relative protein abundances were calculated for all proteins detected. The results are summarized in None of the peptides corresponding to four of the secreted proteins analysed could be detected in any of the strains  and these proteins were not considered in the analysis, leaving 27 proteins. Twelve proteins had undetected peptides in the Δsli-fkbp strains should highlight the effect of FKBP. All the proteins (both Sec and Tat secreted) were significantly affected in the Δsli-fkbp strain with respect to the overproducing strain; the relative expression ratio could not be calculated for proteins not detected in the Δsli-fkbp strain. On the other hand, comparison of pFDTFKBP with the isogenic pFDT strain should tag proteins that may be affected by overexpression of sli-fkbp. Besides the expected difference in Sli-FKBP, nine other secretory proteins displayed a significant difference in their relative abundance when Sli-FKBP was expressed in multi-copy (pFDTFKBP) versus single-copy (pFDT) strains.As expected, Sli-FKBP (SLI29545) showed significant differences in the relative protein abundance among the three strains according to their corresponding genotype. Comparison of the extreme cases pFDTFKBP and ΔThe increments in relative protein abundances observed in the presence of Sli-FKBP suggest that it may have a role in contributing to improve the stability of a wide number of proteins.S. lividans TK21, Sli-DsbA and Sli-DsbC [S. lividans (SLI18490) [The stabilizing effect of Sli-FKBP might be indirect, owing to isomerization of intermediate proteins with post-translocational activity over the final secretory proteins. For this reason, we also included in the MRM/SRM experiment two thiol-disulfide oxidoreductases recently described in Sli-DsbC , a proteSli-DsbC , and theLI18490) . No pept4.S. lividans TK24 secretome [S. lividans, SLIV_29545, which is homologous to SCO1639. The S. coelicolor FKBP-like SCO1639 has been identified as a Tat-dependent lipoprotein [S. lividans TK21 and shown that this protein (which we have named Sli-FKBP) has a PPIase activity that can be inhibited by FK506 .PPIases have been found to be present in almost all sequenced genomes and to be involved in a variety of biological processes ,60–65. Recretome  has beenoprotein , but litby FK506 a,b.sli-fkbp gene increased the transcriptional level of the CssRS operon, suggesting accumulation of misfolded proteins. In addition, absence of sli-fkbp severely affects secretion and activity of Sec-dependent α-amylase , similarly to what has been reported with the prsA mutant [sli-fkbp moderately affects the secretion and activity of Tat-dependent agarase , a protein previously thought to be secreted in a fully folded conformation.The accumulation of secretory misfolded proteins induces a secretion stress response that activates the two-component system, CssRS, which regulates the synthesis of three specific HtrA-like proteases able to degrade misfolded proteins at the late exponential growth phase . As expe-amylase a,b, simiA mutant ,16,66,67 agarase c,d, a prcis conformation may be involved in Sec-dependent α-amylase (at position P350) and Tat-dependent agarase (at positions P127 and P183)  to render the mature active enzymes. Further research is needed to study the implication of Sli-FKBP and these proline residues in their folding and activity.Accordingly, prolyl isomerization to the sli-fkbp gene was propagated in multi-copy in the α-amylase overproducer strain, secretion and activity increased . It has been described that overproduction of PrsA enhances the secretion of α-amylase from Bacillus amyloliquefaciens (AmyQ) [When the ncreased a,b. It hs (AmyQ) , affectiIn the case of α-amylase overproduction, which is expected to form five disulfide bonds, maturation may involve the participation of thiol-disulfide oxidoreductases, as recently published , in addiS. lividans TK21 around the end of the exponential phase of growth [S. lividans TK24 secretome [Despite the Sli-FKBP lipoprotein being predicted to contain a Tat signal peptide  and althf growth , similarecretome . Tat-depecretome . The prec,d). Remarkably, under agarase overproduction conditions, we also observed a shift in its SDS-PAGE mobility that only occurred when Sli-FKBP was overproduced concomitantly (b). Analysis of the peptides produced by trypsin digestion of the protein recovered from the faster migrating band reported identification of most of the agarase protein sequence . The observed band migration does not correspond to the mobility that would be expected if the lack of detection of unrecovered peptides had been due to specific protein degradation. Furthermore, specific protein degradation would conflict with the higher levels of agarase activity measured. Thus, the difference in mobility can hardly be explained just by protein degradation, and seems more likely to be due to gel shifting, a phenomenon previously described where mobility changes in SDS-PAGE gels are explained by changes in the protein conformation that affect its Stokes radius or surface charge. Actually, inspection of the structural models produced for agarase agrees with this observation.Overproduction of Sli-FKBP increased activity of agarase in a proportion greater than what would be expected only by secretion c,d. Remamitantly b. Analyscis, P127trans might keep an active catalytic site without an allosteric agarose-binding site, lacking processive ability with reduced efficiency. This could explain why, in unsaturated Sli-FKBP conditions, agarase overproduction may display lower activity than with Sli-FKBP overproduction .The increase in agarase activity when Sli-FKBP is concomitantly overproduced might be justified by differences in the degree of agarase folding assisted by Sli-FKBP. According to modelling, the form P183The dependency of agarase activity on Sli-FKBP overproduction may provide an interesting model for introducing improvements to the industrial overproduction of other Sli-FKBP-dependent secreted proteins.sli-fkbp strain was used or when sli-fkbp was overexpressed, indicating that Sli-FKBP may have a wide relevance for protein secretion  are involved in protein folding in the periplasm [This is especially striking in the case of proteins secreted through the Tat route. Tat-dependent proteins are very few in comparison with Sec-dependent ones; not all of the verified 27 Tat-dependent proteins  may be p PPIases . In a si PPIases . Unfortulividans . SLI1849omycetes ,69. Togeeriplasm . Further5.S. lividans that may be involved in the correct folding of a wide number of Sec- and Tat-dependent secretory proteins. Our results indicate that, in practical terms and regardless of the route used, when the relative amount of overproduced secretory protein exceeds the capacity of the proteins in charge of procuring them a correctly folded structure, it may be convenient to adjust the level of expression of specific folding enzymes to ensure complete folding and optimize the overproduction of functional homologous or heterologous secretory proteins in S. lividans.We have identified an FKBP-like protein in


In this study, Al-1.00 Mg-0.65 Si-0.24 Cu alloy was solution heat-treated, water-quenched, and then pre-deformed for 5% before aging. The peak hardness and yield strength of the pre-deformed sample with subsequent artificial aging were similar to that of a T6 condition sample. It was also found that the pre-deformation treatment could inhibit the negative influence of natural aging to some degree. After seven days of natural aging, the pre-deformed sample obtained better peak hardness and yield strength upon artificial aging than the sample without pre-deformation. In addition, the pre-deformation treatment could reduce 50% of the artificial aging time to reach the peak aging condition compared with T6 treatment. For the peak aged condition in the pre-deformed sample, transmission electron microscopy (TEM) observation found two types of precipitates exhibited along the dislocations besides the β″ precipitates in the Al matrix. Both precipitates had disordered atomic arrangements on the ordered subcell (Si network). The disordered precipitates occupied a number of Mg and Si atoms, resulting in less β″ precipitates formed during artificial aging at 180 °C. The 6xxx series Al-Mg-Si-Cu alloy is an important series of Al alloys for application as structural materials ,2. PreciEdwards et al.  reportedAfter solution heat treatment, the aluminum materials are usually stored for more than one week at room temperature before being put into further application, during which aluminum undergoes a natural aging process (NA) . PogatscSome studies suggested that adopting deformation after water quenching could affect the precipitation for Al-Mg-Si-Cu alloy. The dislocations that are generated by deformation decreased the activation energy for precipitates and accelerated precipitate reaction . MoreoveUp until now, the precipitation of Al-Mg-Si-Cu alloy after deformation is still the subject of many disputes. In addition, few studies have investigated the effect of deformation on the mechanical properties during subsequent AA, and it is also necessary to identify whether the negative influence of NA can be inhibited by introduced pre-deformation. In the present work, hardness measurement, tensile test, transmission electron microscopy (TEM), HRTEM, and scanning electron microscopy (SEM) analysis were adopted to study the effect of 5% pre-deformation on the mechanical properties and precipitation behavior upon the AA of an Al-Mg-Si-Cu alloy. The correlation between mechanical properties and microstructure was demonstrated, and the influence of pre-deformation on precipitation behavior has also been discussed.The material used in this work is standard AA6061 alloy, which is widely applied in commercial industry with the composition of 1.00 Mg-0.65 Si-0.24 Cu-0.14 Fe-balance Al (wt %). The cast ingots were homogenized, and then went through hot and cold rolling to achieve a 4.0-mm final gauge. The cold rolled sheets were solution heat-treated at 540 °C for 1 h, and then water quenched to room temperature, followed by cold rolling by 5% (pre-deformation treatment) within 10 min. After pre-deformation, the AA process was performed at 180 °C for 0–300 min, either with or without prior natural aging (NA). In the present work, the processes of the samples are listed in The Vickers microhardness of the samples was performed with a 100-g load, and the holding time is 15 s. At least seven indentations were measured to get an average value of hardness. The tensile tests were carried out on the MTS 810 tensile testing machine . The samples for tensile testing had a gauge length of 36 mm, a width of 10 mm, and a thickness of 4 mm. Three tensile samples were tested for each experimental state. 3 in methanol and was kept at temperatures between −25 °C and −20 °C during thinning. The TEM and HRTEM investigations were performed on an FEI Tecnai F20 TEM  operated at 200 kV. The SEM studies were measured by a HITACHI s-4300 microscope  at 20 kV. The TEM specimens were firstly prepared by mechanical polishing and followed by electropolishing. The electrolyte consisted of 33% HNOThe changes in hardness during artificial aging at 180 °C for the samples are displayed in It can be seen in Al zone axes. To study the precipitation behavior of peak artificial aging condition (AA 120 min after pre-deformation) in the PD sample, TEM analyses were selected. All of the micrographs in this study were obtained at <001>Moreover, HRTEM analysis was carried out to investigate the Type 1 and Type 2 precipitates. As shown in The hardness has exhibited a marked increase after seven days of natural aging in the WQ sample shown in a. AccordFor the pre-deformed samples, the hardness of the sample increased after 5% cold rolling b. Howevek0 is the frequency factor, Q* is the activation energy, R is the gas constant, T is the absolute temperature, and f(Y) is only dependent on Y. The results suggest that deformation could significantly decrease the activation energy for precipitates. In summary, the dislocations introduced by pre-deformation are helpful for the formation of precipitates. On the other hand, the amount of solute atoms that are occupied by Type 1 and Type 2 precipitates result in less β′′ precipitates being formed at peak aged condition. Therefore, the pre-deformation treatment could reduce 50% of the AA time to reach the peak aging condition. It is worth pointing out that the artificial aging time to reach peak condition is decreased by 50% in the PD sample. Quainoo and Yannacopoulos  suggesteAfter quenching, 5% pre-deformation cannot increase the peak hardness and yield strength upon subsequent artificial aging compared with the T6 condition. However, the pre-deformation treatment could inhibit the negative influence of natural aging to some degree. After seven days of a natural aging process, the pre-deformed sample has better peak hardness and yield strength upon artificial aging.The sample with 5% pre-deformation treatment reaches the peak artificial aging condition after 120 min, compared to 240 min of the sample without pre-deformation.The pre-deformation treatment could reduce 50% of the artificial aging time to reach the peak aging condition compared with T6 treatment. The dislocations generated by pre-deformation significantly accelerate the process of precipitation during artificial aging.Type 1 and Type 2 precipitates are exhibited along dislocations after pre-deformation, which have disordered atomic arrangements on the ordered subcell (Si network). The disordered precipitates occupied a number of Mg and Si atoms, and result in fewer β" precipitates being formed during subsequent artificial aging.In this work, SEM, TEM, HRTEM, hardness, and tensile measurements have been applied to investigate the effect of 5% pre-deformation on the mechanical properties and precipitation behavior upon AA of an Al-Mg-Si-Cu alloy. The following conclusions are obtained:


The regeneration of neurons is an important goal of neuroscience and clinical medicine. The electrical stimulation of cells is a promising technique to meet this goal. However, its efficiency highly depends on the electrochemical properties of the stimulation electrodes used. This work reports on the preparation and use of a highly electroactive and biocompatible nanoelectrode made from a novel polyaniline functionalized graphene composite. This nanocomposite was prepared using a facile and efficient polymerization-enhanced ball-milling method. It was used to stimulate the growth of PC12 cells under various electrical fields. The enhanced growth of axons and improved wound regeneration of PC12 cells were observed after this treatment, suggesting a promising strategy for neuro traumatology. Neuro traumatology can impact the whole nervous system. It is mainly caused by neural traumata, which itself is caused by traffic accidents, tumor damages, and side effects from neurosurgery . Neural Electrical stimulation (ES) is considered a potentially useful therapeutic for the nervous system and for wound healing ,4. Electinj, i.e., electrochemical capacitance). However, the regular clinical use of ES was thwarted because of the absence of suitable stimulation electrodes with a high Qinj, good biocompatibility, and long-term stability [A fundamental issue in the development of electrically stimulated regeneration of neurons is the correct selection of electrodes that have enough of a safe-charge-injection limit , functionalized with graphene via a polymerization enhanced edge-functionalization ball-milling (EFBM) method. Along with other groups from reports elsewhere ,16, we dThe as-synthesized PANI-G was then applied here to the growth of PC12 cells. PC12 is a cell line derived from the pheochromocytoma of the rat adrenal medulla. PC12 cells are frequently used as a cell model for neuronal studies since PC12 cells can easily differentiate into neuron-like cells . In thisAs described, PANI-G was prepared by ball-milling the mixture of aniline and graphite. The nanomaterials produced were dispersed in Hank’s balanced salt solution  and coated onto a flexible ITO before applying the PANI-G nanoelectrodes to perform an ES a. Due to−1 (due to the C-C stretching of the benzenoid ring), 1330 cm−1 (because of the C=C stretching vibration in the quinonoid ring), 1490 cm−1 (attributed to the C=N stretching vibration), and 990 cm−1 , respectively. As soon as the PANI-G was confirmed, peaks at 1160 and 990 cm−1 were visible, while the bands at 1330 and 1490 cm−1 became overlapped with the characteristic D band and G band of graphene, respectively. The peak at 2700 cm−1 indicates the 2D band of graphene. Raman results confirmed the formation of PANI-G. A Fourier transform infrared spectroscopy (FTIR) of PANI-G  identified the morphology of the as-prepared PANI-G using a tapping mode. As shown in f PANI-G  providedTo improve the application of PANI-G in cell cultures and regeneration, we also tested the biocompatibility of PANI-G with PC12 cells. The electrochemical properties of PANI-G were tested using acyclic voltammogram (CV) of PANI-G in HBSS (pH = 7.3). As a control, pristine graphite powder was treated under identical milling conditions, but without added polyaniline. The milled graphite and the pristine polyaniline were then coated to a glassy carbon (GC) electrode for electrochemical characterization. As shown in In vitro electrical stimulation was applied to PC12 cells attached PANI-G nanoelectrodes as described in the experimental part. As shown in p < 0.01, p < 0.005, Cell axon length was measured as the distance between the edge of the nucleus and the end of the axon . As we cTo better determine ES effects on cell axon growth, stimulation time was tested from 3 days to 7 days. As seen in 3/mm2 in the control to 1.83 × 103/mm2 after ES treatment for 1 h per day, over 5 days. However, the p value of the difference between the control and the ES group was higher than 0.05, indicating the difference was not significant. When ES frequency was increased to 3 h per day, the cell density was measured at 1.81 × 103/mm2. Again, the p value was higher than 0.05. This suggests that the use of ES has no adverse impact on cell activity.Further tests on the impact of ES on the cell density of PC12 cells were performed. PC12 cell nuclei were stained with DAPI blue, . FluoresThe impact of ES on the wound healing ability of PC12 cells was also tested by using a mechanically induced damage model. In a typical test, PC12 cell seeded PANI-G nanoelectrodes were mechanically scratched across their middle part  of the PANI-G is also important for the ES application.During the application of ES, the extracellular region is driven to a more negative potential while the intracellular compartment is driven to a more positive potential. A charge is thus transferred across the cell membranes due to passive cell membrane properties and active ion channels. The QThe ES process parameters used impacted the stimulation effects. Modifiable parameters include the applied potential waveform type, stimulation frequency, stimulation duration, and intensity of the applied electric field. (1) For example, the use of monophasic pulsingstimulation can be effective, but it causes bio-damage during long-term stimulation due to the great negative overpotentialscaused during pulsing. Compared to monophasic pulsing, biphasic pulsing creates lower negative overpotentials. During biphasic pulsing, the first stimulating phase elicits the initiation of an action potential, while the secondreversal phase reverses the direction of electrochemical processes occurring from the first phase. (2) It was observed that 1 h per day of ES was effective in promoting both motor and sensory axon regeneration . StimulaThe function of the nervous system is dependent on the highly specific connections between neurons, achieved by neurite extension to correct targets . The regThe PANI-G was prepared via a polymerization-enhanced edge-functional ball-milling method. In a typical preparation, 10 mg graphite powders  were placed in 10 µL aniline . The mixture was milled in a planetary ball-milling machine  set at a spin rate of 400 to 500 rpm for 6 to 8 h. The formed material was then transferred to a centrifuge tube containing 50 mL dehydrated alcohol. It was then centrifuged at 1000 rpm for 10 min to remove any remaining graphite and large sized-particles left in the precipitate. The supernatant was collected and re-centrifuged at 6000 rpm for 10 min to remove the remaining monomer left in the supernatant. The as-processed precipitate was then placed in a dialysis bag . It was soaked in dehydrated alcohol for 72 h to further remove any remaining impurities. The final PANI-G materials were dispersed in HBSS  before testing it on biological samples.The physiochemical properties of the as-prepared PANI-G nanosheets were characterized using Raman spectroscopy  and atomic force microscopy . The electrochemical properties and ES were performed using a CHI 760D electrochemical work station. Cyclic voltammetry of nanomaterials was carried out on a PANI-G coated glassy carbon (GC) electrode by dropping PANI-G onto a GC electrode (3 mm i.d.) and allowing it to be dried in air.2.Indium tin oxide (ITO) coated conducting film  was used as the supporting substrate for the PANI-G membrane. The ITO film was pre-treated by applying a radio-frequency glow discharge plasma  in an ox2 in a humid atmosphere. The PANI-G/ITO membrane was cut into circular pieces to fit into the culture wells. The membrane was sterilized by a 75% ethanol solution and exposed to ultraviolet light for 1 h, prior to its use in cell culturing. The biocompatibility of PANI-G was tested using a Cell Counting Kit-8  assay. This assay measures cell viability in cell proliferation and cytotoxicity using a sensitive colorimetric reaction. Dehydrogenase activities within cells reduce a highly water-soluble tetrazolium salt, WST-8 (developed by Dojindo). This forms a yellow-colored formazan dye soluble in the culture media. The amount of this dissolved dye can be used to count the number of living cells, which is proportional to the amount of dissolved dye. In this work, PC12 cells were seeded in a 96-well plate at a density of 5000 cells/well. Cells were pre-incubated for 24 h in a humidified incubator . PANI-G at various concentrations was added to the cell wells and co-cultured with nanomaterials over 24 to 72 h, before washing the cells with HBSS. A total of 10 µL CCK-8 solution was added into each well. The culture plate was then placed in the incubator over 0.5 to 2 h. The culture media absorbance was measured at 450 nm with a microplate reader .PC12 cells  were cultured in RPMI 1640 Medium  supplemented with 10% heat-inactivated qualified fetal bovine serum  and 0.2% gentamicin solution . The cells were then incubated at 37 °C with 5% CO3/mm2 and pre-cultured for 24 h. A platinum (Pt) electrode (ϕ = 0.5 mm) was contacted with the PANI-G/ITO electrode, while another Pt electrode was placed in the cell culture medium. The distance between these electrodes remained at 1 cm. An electrical field was applied to these cells using a double-pulsed potential chronoamperometry. A schematic set-up of the electrical stimulation is shown in PC12 cells were seeded on the PANI-G/ITO electrode to a density of 0.25 × 102. The properties of PC12 cells were measured after ES. As a negative control, PC12 cells were also cultured on pristine ITO film (that did not have a PANI-G coating) before testing with ES. Negative controls were also tested. PC12 cells were cultured in plates with no electrical stimulation.A forward 500 mV/cm potential and a reverse −500 mV/cm potential were applied. ES was carried out for 1 or 3 h each day. The stimulation was run for 3, 5, and 7 days, respectively. After this treatment, the medium was refreshed immediately to avoid its contamination. PC12 cells were then maintained in the incubator at 37 °C under 5% COPC12 cells attached to PANI-G membranes were fixed by applying 4% paraformaldehyde  for 10 min and then permeabilized with 0.1% Triton-X 100 solution  for 5 min. A total of 1 µg/mL 4’,6-diamidino-2-phenylindole (DAPI) was used to visualize nuclei. Superfluous dye stuff was washed away using PBS. A drop of antifade medium  was added to the cells, which was sealed with transparent nail polish. Images of samples were captured using fluorescent microscopy . The axon length of the PC12 cells was measured along the linear distance between the brink of a nucleus and the tip of an axon. The mean axon length was calculated by averaging the length of 300 measurements in each sample. These averages were then statistically analyzed across different samples. The mean cell density was calculated by imaging 3 fields in each sample and statistically analyzed. The cell density was countered using DAPI-stained fluorescence images that were analyzed with an Image-J software.t-test for each variable tested was calculated by comparing the wound healing ability of the ES group to a negative control. Statistical differences were calculated using a SPSS 16.0 software . A * p < 0.05 was predetermined as significant difference in these calculations. A ** p < 0.01 indicates a higher significance and a *** p < 0.005 represents a very high significance.A one-way analysis of variance (ANOVA) and post-hoc test statistically compared the changes in the axon length and cell density after electrical stimulation . A t-tesThis work reported a facile and efficient ES system, based on the PANI-G nanoelectrodes, to regenerate PC12 nerve cells. A high electroactive and biocompatible PANI-G nanomaterial was prepared via a polymerization-enhanced EFBM method. ES studies on PC12 cells attached to the PANI-G nanoelectrodes showed that ES can play a role in promoting PC12 cells regeneration. A 60% increase in axon length, with no adverse impact on the cell density and enhanced wound regeneration ability of PC12 cells after ES, was observed. The novel stimulation nanoelectrodes reported here provide a new choice for ES stimulation, facilitating the clinical application of ES in various fields, such as neuro traumatology.


Little is known about the modulation of cortical excitability in the prefrontal cortex during fear processing in humans. Here, we aimed to transiently modulate and test the cortical excitability during fear processing using transcranial magnetic stimulation (TMS) and brain oscillations in theta and alpha frequency bands with electroencephalography (EEG).We conducted two separate experiments (no-TMS and TMS). In the no-TMS experiment, EEG recordings were performed during the instructed fear paradigm in which a visual cue (CS+) was paired with an aversive unconditioned stimulus (electric shock), while the other visual cue was unpaired (CS-). In the TMS experiment, in addition the TMS was applied on the right dorsomedial prefrontal cortex (dmPFC). The participants also underwent structural MRI (magnetic resonance imaging) scanning and were assigned pseudo-randomly to both experiments, such that age and gender were matched. The cortical excitability was evaluated by time-frequency analysis and functional connectivity with weighted phase lag index (WPLI). We further linked the excitability patterns with markers of stress coping capability.After visual cue onset, we found increased theta power in the frontal lobe and decreased alpha power in the occipital lobe during CS+ relative to CS- trials. TMS of dmPFC increased theta power in the frontal lobe and reduced alpha power in the occipital lobe during CS+. The TMS pulse increased the information flow from the sensorimotor region to the prefrontal and occipital regions in the theta and alpha bands, respectively during CS+ compared to CS-. Pre-stimulation frontal theta power (0.75–1 s) predicted the magnitude of frontal theta power changes after stimulation (1–1.25 s). Finally, the increased frontal theta power during CS+ compared to CS- was positively correlated with stress coping behavior.Our results show that TMS over dmPFC transiently modulated the regional cortical excitability and the fronto-occipital information flows during fear processing, while the pre-stimulation frontal theta power determined the strength of achieved effects. The frontal theta power may serve as a biomarker for fear processing and stress-coping responses in individuals and could be clinically tested in mental disorders. Fear is an emotional response that is triggered in the brain in anticipation of a potentially dangerous event . InstrucAccumulating evidence indicates that instructions about the CS+/US contingency evoke effects on the neural activity of a distributed network of brain regions, namely the amygdala, the cingulate cortex, the insula, hippocampus and prefrontal cortices, among which the dorsomedial prefrontal cortex (dmPFC) plays an important role in fear processing by dynamically regulating excitability . A recenIn a previous study, we analyzed the TMS-evoked potentials (TEP) measured with EEG and TMS over the right dmPFC during an instructed fear paradigm . We shown = 19, 9 males, mean age 27.4 ± 4.32 years, TMS experiment: n = 19, 10 males, mean age 28.6 ± 6.64 years) were included in our study. All participants had two visits to the lab. During the first visit, MRI data was acquired, whereas, at the second visit, an instructed fear paradigm (no-TMS experiment) or an instructed fear paradigm together with TMS (TMS experiment) was performed. Participants were assigned to one of the two experiments pseudo-randomly, such that age and gender were matched. The TMS experiment was conducted after completing the no-TMS experiment.Thirty-eight healthy subjects  equipped with a 32-channel head coil at the Neuroimaging Center (NIC) in Mainz, Germany. A magnetization-prepared rapid gradient-echo (MP-RAGE) sequence was used .1 in Matlab 2006b (MathWorks). First, the participant was asked to sit on a chair, and an electric shock was applied to the dorsal part of the left hand using a surface electrode that was connected to a DS7A electrical stimulator (Digitimer). Then, the participant was requested to rate the amount of pain perceived on a scale from 0 (no pain) to 10 (intense pain). An electric shock intensity corresponding to a pain level of 7 was employed during the experiment  through a figure-of-eight coil with internal wing diameter of 70 mm. First, the resting motor threshold (RMT) was determined as the minimum stimulus intensity required to elicit motor evoked potentials of amplitude 50 μV in 5 out of 10 consecutive trials at rest   . We used2 and MATLAB R2015b (The MathWorks). The EEG data was cut in epochs of 7 s within the time interval of -2 to 5 s from the onset of the visual cue  that is a measure of global cortical excitability. Afterward, we assessed the local cortical excitability specifically in the frontal and occipital lobes by estimating the power in theta and alpha frequency bands, respectively. In addition, we also investigated the direction of information flow in these frequency bands with the weighted phase lag index (WPLI) method. The WPLI works based on phase synchronization and is less sensitive to uncorrelated noise sources and has increased statistical power to detect the alterations in phase compared to other methods, such as phase lag index (PLI) . FurtherAt the end of the experiment, all the subjects were asked to rate their perceived level of fear during the experiment, for both CS+ and CS- independently, on a scale from 0% (not fearful/safe) to 100% (very fearful). Heart rate was estimated from the EEG signals using the extended version of the ICA algorithm  based on-1, give the projection strengths of the respective components onto the scalp sensors.For EEG analysis, the EEG signals recorded at the 256 electrodes represent the rows of the sensor input matrix y for the ICA component extraction, the rows of the output data matrix v = Xy are time courses of activation of the ICA components, and the columns of the inverse matrix, X-1v′, where v′ is the matrix of activation waveforms, v, with rows representing cardiac artefactual sources which are then extracted for further estimations from each participant. In total for the no-TMS experiment, we concatenated the 36 CS+ trials to have 180 s and 36 CS- trials to have 180 s. For the TMS experiment, we concatenated the 36 CS+ trials to have 180 s and 36 CS- trials to have 180 s.In general, and unlike principal component analysis (PCA), the component time courses of activation will be non-orthogonal. Corrected EEG signals can then be derived as y′ = (X)The GMFP is an index of distinctive global cortical excitability and also reflects the synchronous activity across observations in response to a specific stimulus , and wasT = 3 cycles = 3/f). The TFRs were computed for the time range of -0.25 to 2 s for frequencies from 4 to 13 Hz. The time-frequency grand averages across subjects were then computed for both CS+ and CS- conditions separately. The relative baseline correction was applied by dividing the post-visual cue onset power with pre-visual cue onset power (-0.25 to 0 s). In this study, the theta and alpha oscillations were investigated in the frontal and occipital lobe, respectively. The electrodes corresponding to the frontal and occipital lobes are shown  of power. The time-frequency analysis was performed using a multitaper method . The EEGre shown .The WPLI is a functional connectivity measure and evaluates the distribution of phase angle differences between two signals . SpecifiAll subjects completed a BRS questionnaire . The BRS3). Post hoc tests were performed if the F-values were significant (p < 0.05) with the Bonferroni correction method, unless otherwise explicitly specified. To study the differences between the stimulus conditions (CS+ and CS-) in behavioral fear ratings and heart rate, we conducted paired t-tests. To examine the global cortical excitability dynamics over time windows during fear processing, a one-way repeated measures analysis of variance (rmANOVA) was conducted with the factor Time , and a dependent measure of GMFP difference between stimulus conditions. To study the differences between stimulus conditions and also across temporal windows, a two-way rmANOVA was run including two factors, Condition (2 levels: CS+ and CS-), and Time, with the dependent measures for separate variables, power in the theta and in the alpha band at the frontal and occipital lobe electrodes  = 17.42; p < 0.001; TMS experiment: t(36) = 18.47; p < 0.001), as shown in t(36) = 6.26; p < 0.001; TMS experiment: t(36) = 5.98; p < 0.001)  for the CS+ and CS- conditions in the no-TMS experiment were 50.2 + 26.9 and 2.6 + 8 and in the TMS experiment 50.5 + 17.5 and 6.8 + 8.8, respectively. The reported fear ratings evidenced well induced fear in the participants during the CS+ condition in comparison to the CS- condition in both experiments (no-TMS experiment: < 0.001) .F = 4015.7, p < 0.001). Post hoc testing showed that the difference of GMFP increased in all the time windows (0–2 s) compared to the baseline time window . Furthermore, the GMFP difference reduced in time windows (1–1.75 s) relative to (0.75–1 s) window . We repeated the same analysis for TMS experiment  = 7404.1, p < 0.001). The post hoc comparisons showed that the GMFP difference increased significantly in the time windows  compared to the (-0.25–0 s) time window . Moreover, the GMFP difference increased significantly in the time windows (1.25–1.5 and 1.75–2 s) compared to the time window  after single-pulse TMS.periment . One-wayF = 619.7, p < 0.001) and a significant main effect for the factor Time  = 240.1, p < 0.001). The interaction between the factors was also significant  = 1626.2, p < 0.001). The post hoc comparisons revealed that in the no-TMS experiment, the theta power was higher during CS+ in all the temporal windows (0–2 s) compared to the baseline time window  (p < 0.05). Furthermore, the theta power was reduced during CS+ in the time windows (1–2 s) compared to the (0.75–1 s) time window. And also, the frontal theta power was decreased for CS- in the window (1–1.25 s) compared to the time window (0.75–1 s). In the TMS experiment, frontal theta power was higher during CS+ in all the temporal windows (0–2 s) relative to baseline window (-0.25–0 s), while for CS- it was decreased in the time window (0–0.25 s) compared to baseline window  (p < 0.05).The grand averaged theta power for the time (-0.25–2 s) with respect to stimulus onset across frontal lobe electrodes are presented separately for different stimuli (CS+ and CS-) in the no-TMS experiment  and TMS  < 0.05) . The fro < 0.05) . As a reF = 4518.2, p < 0.001), significant main effect for the factor Time  = 2391.2, p < 0.001) and the interaction between factors was also significant  = 2391.2, p < 0.001). The post hoc tests showed a significant decrease in occipital alpha power during CS+ in the time window (0–1.25 s) but an increase for CS- in the (0–2 s) compared to a baseline time window  (p < 0.05). In the TMS experiment, the occipital alpha power was decreased for CS+ while increased for CS- in all the time windows (0–2 s) compared to a baseline time window  (p < 0.05). Further to providing surface topography of the oscillatory power changes, we performed comparisons between stimulus conditions in the theta and alpha frequency bands. In both experiments, statistical comparison of stimulus conditions CS+ and CS- revealed an increase in theta power and a decrease of alpha power in the latency range 0.15–0.45 s   . In the  < 0.05) . Due to  < 0.05) .t18 > 2.2, p < 0.05)    , while the premotor regions received information from prefrontal regions in the first temporal window (0–0.25 s) after visual cue onset in the theta band  . In the  < 0.05) . In the  < 0.05) . Moreove < 0.05) .F = 66.6, p < 0.001), explaining up to 93% of the oscillatory activity increase after TMS stimulation. These effects were only significant in the time window 0.75–1 s . The linear regression showed no significant relationship in alpha power between time windows before (0–1 s) and after (1–1.25 s) TMS pulse delivery.A linear regression was performed to predict theta power increase after TMS pulse delivery in the time window (1–1.25 s). The results showed a high prediction power from pre-TMS pulse activity . In the TMS experiment, theta power correlated with BRS across all subjects in the time windows (0.15–0.45 and 1–1.25 s)  suggesting that the increases in the theta power during CS+ relative to CS- are related to the individual coping abilities.In the no-TMS experiment, the correlation between theta power (0.15–0.45 s) and BRS showed a positive statistical trend  predicted modulatory effects following 10 Hz prefrontal repetitive stimulation . SimilarWe found that people with higher BRS scores showed higher frontal theta power during fear processing. This result suggests that resilient mechanisms play a key role in modulating fear processing. We also showed that people with low BRS score were less susceptible to the modulatory effects of dmPFC-TMS stimulation, suggesting that preserved ability for coping with aversive situations is directly related to specific patterns of cortical excitation and communication within the regions forming fear network. It will be of interest to evaluate this possibility in people suffering with anxiety-related disorders in future studies.In this study, dmPFC localization was performed with a neuronavigation system based on individual T1-weighted MRI data and coordinates obtained from the previous study . DetermiIn summary, our results provide insight into the dynamics of cortical excitability modulation and functional connectivity during fear processing, while the patterns of pre-stimulation frontal theta power determine the magnitude of effects induced by TMS stimulation. Moreover, frontal theta power is clearly related to an individual’s ability to cope with challenging situations, leaving those individuals with low coping abilities more vulnerable to functional failure in face of adversity.VC contributed to data acquisition, data analysis, and manuscript writing. NK, TB, and SvG contributed to data acquisition. GG-E contributed to data discussion and manuscript writing. MM and SG contributed to experimental design, data analysis, and revision of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Melatonin and nitric oxide (NO) are involved in orofacial signal processing in the trigeminal sensory system. The aim of the present study was to examine the distribution of melatonin 1a-receptor (MT1) and its colocalization with nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) in the spinal trigeminal nucleus (STN), the trigeminal ganglion (TG), and the mesencephalic trigeminal nucleus (MTN) in the rat, using histochemistry and immunohistochemistry. Our results show that MT1-positive neurons are widely distributed in the TG and the subnucleus caudalis of the STN. Furthermore, we found that MT1 colocalizes with NADPH-d throughout the TG and MTN, most extensively in the TG. The distribution pattern of MT1 and its colocalization with NADPH-d indicate that melatonin might play an important role in the trigeminal sensory system, which could be responsible for the regulation of NO levels. Orofacial sensory signals are conducted to the central nervous system by the trigeminal sensory system, which comprises two populations of primary afferent neurons. The majority are located in the trigeminal ganglion (TG), conveying information from mechanoreceptors, thermoreceptors, and nociceptors in the orofacial region. The remainder are situated in the mesencephalic trigeminal nucleus (MTN) in the central nervous system and are involved in proprioception in the masticatory and extrinsic ocular muscles and a subset of periodontal ligament fibers . The secMelatonin is a functionally versatile neurohormone, produced mainly in the pineal gland in a clear circadian manner, and has a range of activities, including circadian rhythm control and sleep regulation, as well as exerting anti-oxidant and anti-nociceptive effects . It is wNitric oxide (NO) is a neuronal chemical messenger, remarkable for its chemical identity, wide distribution, and broad spectrum of effects . NO is sMelatonin and its receptors are believed to play important roles in nociception and pain modulation . PreviouFive adult female Sprague-Dawley rats (body weight 180–220 g) were used. All of the experimental procedures were approved by the Ethical Review Committee of Guanghua School of Stomatology, Hospital of Stomatology, Institute of Stomatological Research, Sun Yat-Sen University. Animals were deeply anesthetized with 10% chloral hydrate  and perfused intravascularly with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brainstem and TG were excised, postfixed in the same fixative solution for 6 h, and then stored overnight in 30% sucrose in phosphate buffer at 4 °C until they sank. The brainstem was cut at the level of the Vc (11.30–15.60 mm posterior to bregma) and MTN (9.04–9.76 mm posterior to bregma according to the rat brain atlas). Horizontal sections (25 μm) were cut through ganglia and transverse sections (25 μm) through the brainstem on a cryostat . Three series of sections were collected in 0.01 M phosphate-buffered saline (PBS).2O2) for 20 min, Ultra V Block  was applied to block nonspecific background staining for 20 min. Sections were then incubated with polyclonal rabbit anti-MT1 serum  at 4 °C for 18–24 h. For TG and MTN sections, immunofluorescence was used to detect MT1. TG and MTN sections were incubated with TMRITC-conjugated donkey anti-rabbit IgG  at room temperature for 1 h in the dark. Subsequently, the sections were mounted in weak natural light with glycerol buffer and stored at 4 °C in the dark overnight. STN sections were incubated with biotinylated goat anti-polyvalent . After incubation in streptavidin peroxidase , the immunoreactive product was visualized by incubation with diaminobenzidine. Slices were mounted on glass slides, air-dried, gradient-ethanol dehydrated, xylene-cleared , and mounted with neutral gum. For double-labeling of MT1 and NADPH-d, NADPH-d histochemistry was followed by MT1 immunofluorescence/immunohistochemistry staining in the same tissue section. All dilutions and washes between steps were performed using PBS and all steps were carried out at room temperature unless otherwise specified. NADPH-d and MT1 negative control sections were incubated as described but omitting β-NADPH or primary antibodies, resulting in an absence of staining.The first and second sets of sections for NADPH-d were stained by using 0.1 M Tris buffer (pH 8.0) containing one mM β-NADPH  and 0.1 mM nitroblue tetrazolium  with 0.3% Triton X-100. They were kept at 37 °C for 1–2 h. Then the second and the third set were processed for MT1 immunohistochemistry. Following blocking of endogenous peroxidase activity with methanol containing 0.3% hydrogen peroxide (H2), medium , and large (>150 μm2). The percentage of coexpression was determined from three sections per rat.After NADPH-d histochemistry and MT1 immunohistochemistry the sections were examined under a light or fluorescence microscope  equipped with a CCD camera . Images were processed using Axio Vision 4.5 imaging software  and Image-Pro Plus software . Three sections from each series were randomly selected and images were generated using a 200× magnification objective. NADPH-d- and MT1-positive neurons in the TG were subsequently categorized by size as small (<85 μmNicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d)-positive neurons were observed in the TG  and 1B. Many MT1-positive neurons were observed in the TG  and 1D. Almost all of the NADPH-d-positive neurons in the TG were double-labeled with MT1  and 1F. Nicotinamide adenine dinucleotide phosphate-diaphorase-positive neurons were observed in the MTN  and 2B. Most NADPH-d-positive neurons in the MTN coexpressed MT1  and 2F. Nicotinamide adenine dinucleotide phosphate-diaphorase-positive cells and fibers were distributed throughout the STN  and 4B. MT1-positive neurons were distributed extensively throughout the STN  and 4D. There was no clearly double-labeled neuron for MT1 and NADPH-d in the STN, and only a few neurons were weakly double-stained  and 4F. Melatonin receptors have been studied for several decades. Studies using autoradiography , in situHowever, the tissue and cellular distribution of MT1 in the trigeminal sensory system is unclear. The present study demonstrates that MT1 is expressed widely in the TG, Vc, and MTN. A previous study from our research group revealed a significant upregulation of NADPH-d in the TG and downregulation of MT1 in the Vc during the early stages of orofacial inflammatory pain . Also, WThe Vc has many anatomical and functional similarities with the spinal dorsal horn; it has a laminated structure and contains several morphologically distinct neuronal types that are generally comparable with those described in the spinal dorsal horn . The TG To date, three distinct isoforms of NOS have been identified, namely, constitutive neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) . In the Other studies support a pronociceptive effect of NO in orofacial pain processing. Primary sensory fibers innervating the orofacial region are derived from neurons of the TG and MTN . The TG + channel pathway. As mentioned above, melatonin exerts its effect on spinal cord nociceptive transmission through the NMDA receptor-dependent pathway  . To date pathway . An NMDA pathway . Further pathway . Togethe−9 M, melatonin transiently increases synthesis of the nNOS protein and NO oxidation products in cultured HaCaT cells; these results are similar to those of Few studies have examined the relationship between melatonin and NO in the orofacial sensory system, although some indirect evidence suggests that there is an interaction between them. NO, a potential source of free radicals, may play an important role in inflammatory and neuropathic pain . MelatonIn brief, MT1 is widely expressed in the trigeminal sensory system, particularly in the TG and Vc. Furthermore, colocalization with NADPH-d was observed, most extensively in the TG and MTN, suggesting that an important interaction exists between melatonin and NO in these nuclei. Although the physiological roles of melatonin and NO in the nervous system are not yet fully elucidated, our results provide morphological evidence for the involvement of melatonin in orofacial sensory modulation by the regulation of NO levels.10.7717/peerj.6877/supp-1Supplemental Information 1Click here for additional data file.


The aim of this study was to evaluate the use of glabellar vertical line (GVL) as the anteroposterior maxillary position goal. A cross-sectional study was conducted assessing 129 participants (20.21 ± 1.99 years): 67 women (20.16 ± 1.99 years), and 62 males (20.26 ± 2.06 years). The facial profile photographs were taken with a posed smile in natural head position. The linear distance from the most facial convexity of the upper central incisor (FA) to the goal anterior-limit line (GALL) and also from FA to GVL were measured and compared. Wilcoxon signed-ranks test was applied. To determine the correlation between the distances, Spearman’s correlation coefficient was used. Simple and multiple linear regression were also performed. p< 0.01) was identified. The FA-GVL distance explains almost all the total variation of FA-GALL . The FA-GALL distance can be predicted by using the formula: FA-GALL=0.5+0.9*(FA-GVL). The GALL-GVL separation was 0.54 ± 1.14 mm (95%CI: 0.34-0.74). A strong correlation between FA-GALL and FA-GVL distances (Spearman’s rho=0.983 [95%CI: 0.976-0.988], These findings suggest that GVL could be used as an easier-to-use treatment goal to determine the maxillary anteroposterior position, compared to GALL, to improve facial harmony profile goals in cases where the maxillary incisors are properly positioned anteroposteriorly. In 1900, Edward H. Angle-Andrews16The correct maxillary anteroposterior position occurs when the FA point of the upper central incisor is located between FALL and GVL, ideally coinciding with GALL, which becomes a treatment goal. When FA is forward GVL, there is maxillary protrusion.Of the two distances, FA-GALL and FA-GVL, the first one corresponds to the gold standard. Dr. Andrews considered GALL as the ideal aesthetic anteroposterior location of the maxilla.Thereby, the purpose of this study was to evaluate the mean differences and the correlation between FA-GALL and FA-GVL distances. The alternative hypothesis was that the FA-GALL and FA-GVL distances are similar and correlated.Ethics and protocol was approved by the Stomatology Permanent Research Committee of the Antenor Orrego Private University . The ethical approval number was 12602014-FMEHU-UPAO.This study counted with a sample of 129 participants (20.21 ± 1.99 years), 67 women (20.16 ± 1.99 years) and 62 males (20.26 ± 2.06 years). The sample was constituted by volunteer students of a local university from Trujillo-Peru, who met the selection criteria. All participants signed an informed consent. The sample size was calculated using the correlation found between scores of the FA-GALL and FA-GVL distances reported from a pilot study. A statistical power of 80% and a confidence level of 95% were considered. Inclusion criteria were: male or female participant, natural dentition, no missing anterior teeth, any type of malocclusion but with the upper maxillary central incisors clinically considered in an ideal position, with favorable buccolingual inclination related to the occlusal plane.Trichion (T), Superion (S), Glabella (G) and the FFA point of the forehead were identified on the midsagittal plane of the head, as described by Andrews.,The facial profile photographs were taken with a posed smile in natural head position, standardized as a position of the head in an upright head posture with the eyes focusing a point in the distance, at eye level.On each printed standardized photograph, the forehead inclination (FI) was traced to determine GALL, as proposed by Andrews.On each printed photograph, the horizontal linear separation in millimeters from the FA point to the GALL line was measured and it was considered as the FA-GALL distance (control group). Similarly, the horizontal linear separation in millimeters from the FA point to the GVL line was also measured, and considered as the FA-GVL distance (study group).To evaluate the method error, measurements in 10 photographs not considered in the final study were assessed. The forehead identification and the FA-GALL and FA-GVL measurements were carried out by the same researcher twice  in order to evaluate intraexaminer reliability. To assess the interexaminer reliability, the same cases were evaluated by another researcher .Data were processed using the statistical software Stata v. 13 . Means, standard deviations, and the 95% confidence interval values were calculated. The agreement between the observations of the type of forehead was performed by using the weighted kappa coefficient. Tracings were evaluated by the intraclass correlation coefficient test. Before making any comparisons, compliance with the assumptions of normality and homogeneity of variances with Shapiro-Wilk and Levene’s test were evaluated. The FA-GALL group had normal distribution, but the FA-GVL group and the GALL-GVL were not normally distributed; therefore, Wilcoxon signed-ranks test was applied. To determine the correlation between FA-GALL and FA-GVL distances, Spearman’s correlation coefficient was used. A simple linear regression analysis was used to evaluate the possibility of predicting the FA-GALL distance using the FA-GVL distance. An analysis of multiple linear regression, in which age, sex, and FI were included as factors, was also performed. Statistical significance was set at 5% in all tests.Reliability was considered adequate. High concordance with values greater than 0.836 were found .p< 0.01) (The mean difference between FA-GALL and FA-GVL (GALL-GVL distance) in the total sample was 0.54±1.14 mm; in females was 0.64±1.25 mm; in males was 0.44±0.99 mm; in the 18- to 20-year-old group was 0.56±1.13 mm; and in the 22- to 24-year-old group was 0.51±1.16 mm. Comparing the distances between FA-GALL and FA-GVL, it was found statistically significant differences in all cases (p< 0.01) .p< 0.01), with correlation coefficients of 0.983 (95% CI: 0.976-0.988) in the total sample, 0.981 (95% CI: 0.969-0.988) in females, 0.993 in males (95% CI: 0.972-0.990), 0.980 (95% CI: 0.969-0.987) in the 18- to 20-year-old group, and 0.986 (95% CI: 0.975-0.993) in the 22- to 24-year-old group . By multiple regression analysis, it was observed that the GALL-GVL distance was not associated with sex, age, and the type of forehead ], by means of which the FA-GALL distance could be predicted by knowing FA-GVL, since this latter distance would be easier to identify . The coefficient of determination was relatively high as it will explain around 100% (95.84%) of the total variance.Ideally, GALL is expected to be located between FFA and glabella (between FALL and GVL), which has been the starting point for various studies. Cao et al.,-,For Element II diagnosis, it is necessary to locate maxillary incisor anteroposterior position in millimeters relative the FALL line, to know how much movement is necessary to correct its location regarding the GALL line. This measurement is proposed to be performed clinically.The findings of this study can be incorporated into routine orthodontic and orthognathic records, diagnosis, and treatment planning. Both the orthodontist and the orthognathic surgeon can perform the evaluation of the anteroposterior maxillary position analyzing the facial profile in smile, in order to observe the closeness of the upper incisors to the GVL, which could guide the clinician in the final decision of where to locate the maxilla at the end of the treatment. As AndrewsThese findings suggest that GVL could be used as an easy-to-use treatment goal to determine the maxillary anteroposterior position, to improve facial harmony profile goals in cases where the maxillary incisors are properly positioned anteroposteriorly.